User:Jaime B. Hutchison/Sandbox 1



This is the amphiphysin BAR domain from Drosophila melanogaster. The biologically active form of this protein is proposed to be a homodimer.

We would like to make fluorescent mutants of this protein for microscopy applications. One way to do this is by attaching maleimide dye to Cys amino acids present in the protein. The homodimer has 4 native cysteine (Cys) amino acids. (Cys is orange .) Before designing the mutant we need to check that there are Cys sites available on the surface for maleimide attachment. Because Cys 82 looks to be accessible and also because the placement of Cys 66 looks like attaching a fluorophore to it may interfere with membrane binding we will mutate Cys 66. This will be done for each monomer, Cys 66 in chain A and B. The mutation will replace the cysteines at residue 66 with alanines (Ala). (Ala is purple .) Each of the mutated monomers will now have only one binding site at Cys 82. The final mutated dimer with Cys 66 replaced with Ala and Cys 82 ready for fluorophore binding is shown here.